Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for rapid identification of micro-organisms in the routine clinical microbiology laboratory.

The study evaluates the utility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) Vitek MS for identification of microorganisms in the routine clinical microbiology laboratory. From May 2013 to April 2014, microbial isolates recovered from various clinical samples were identified by Vitek MS. In case of failure to identify by Vitek MS, the isolate was identified using the Vitek 2 system (bioMerieux, France) and serotyping wherever applicable or otherwise by nucleic acid-mediated methods. All the moulds were identified by Lactophenol blue mounts, and mycobacterial isolates were identified by molecular identification systems including AccuProbe (bioMerieux, France) or GenoType Mycobacterium CM (Hain Lifescience, Germany). Out of the 12,003 isolates, the Vitek MS gave a good overall ID at the genus and or species level up to 97.7% for bacterial isolates, 92.8% for yeasts and 80% for filamentous fungi. Of the 26 mycobacteria tested, only 42.3% could be identified using the Saramis RUO (Research Use Only) database. VITEK MS could not identify 34 of the 35 yeast isolates identified as C. haemulonii by Vitek 2. Subsequently, 17 of these isolates were identified as Candida auris (not present in the Vitek MS database) by 18S rRNA sequencing. Using these strains, an in-house superspectrum of C. auris was created in the VITEK MS database. Use of MALDI-TOF MS allows a rapid identification of aerobic bacteria and yeasts in clinical practice. However, improved sample extraction protocols and database upgrades with inclusion of locally representative strains is required, especially for moulds.

Microbial identification and automated antibiotic susceptibility testing directly from positive blood cultures using MALDI-TOF MS and VITEK 2.

The study addresses the utility of Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight mass spectrometry (MALDI-TOF MS) using VITEK MS and the VITEK 2 antimicrobial susceptibility testing (AST) system for direct identification (ID) and timely AST from positive blood culture bottles using a lysis-filtration method (LFM). Between July and December 2014, a total of 140 non-duplicate mono-microbial blood cultures were processed. An aliquot of positive blood culture broth was incubated with lysis buffer before the bacteria were filtered and washed. Micro-organisms recovered from the filter were first identified using VITEK MS and its suspension was used for direct AST by VITEK 2 once the ID was known. Direct ID and AST results were compared with classical methods using solid growth. Out of the 140 bottles tested, VITEK MS resulted in 70.7 % correct identification to the genus and/ or species level. For the 103 bottles where identification was possible, there was agreement in 97 samples (94.17 %) with classical culture. Compared to the routine method, the direct AST resulted in category agreement in 860 (96.5 %) of 891 bacteria-antimicrobial agent combinations tested. The results of direct ID and AST were available 16.1 hours before those of the standard approach on average. The combined use of VITEK MS and VITEK 2 directly on samples from positive blood culture bottles using a LFM technique can result in rapid and reliable ID and AST results in blood stream infections to result in early institution of targeted treatment. The combination of LFM and AST using VITEK 2 was found to expedite AST more reliably.

Pulmonary coccidiomycosis in New Delhi, India.

Coccidioidomycosis is an endemic disease of the western hemisphere. In cases occurring in non-endemic areas, eliciting a history of exposure as well as a high index of suspicion is imperative for timely and accurate diagnosis. In this case, a 65-year-old male presented to our hospital with fever, dry cough and malaise, and on X-ray chest, was found to have a lower lobe consolidation left lung with nodular lesions in both lungs and necrotic mediastinal lymphadenopathy. He lived in Arizona, USA, for 6 months before admission. Pulmonary coccidioidomycosis was confirmed by the isolation of Coccidioides spp. in pure culture from both broncho-alveolar lavage and lung biopsy specimens. The identity of the isolate was confirmed as C. posadasii by gene sequencing. The patient improved after being treated with fluconazole.

Surveillance of multidrug resistant organisms in tertiary care hospital in Delhi, India.

Surveillance of multi drug resistant organisms in a health care setting is a necessity to have optimum treatment out come and less of treatment failures. Once any health care setting gets colonized with multi drug resistant organisms, it is very difficult to decontaminate the environment. On review of our data, for 12 months of year 2008 the prevalence of difficult to treat organisms with poor clinical outcome especially in ICUs have been identified. The need for surveillance, prescription auditing & computer assisted retrieval of data has been emphasized and discussed in detail with respect to various high quality samples like blood, urine, respiratory, pus and sterile body fluids. The prevalence of MRSA & VRE has been documented 30 to 40% and 10%, respectively. Over all prevalence of penicillin intermediate resistant Streptococcus pneumoniae was found to be 9.52%. ESBL, AmpC, and Carbapenemase producing organisms were found to be 40 to 60%, 70 to 80% & 2 to 80% respectively in various multi drug resistant organisms like E. coli, Klebsiella spp., Pseudomonas spp. and Acinetobacter spp. 8% Pseudomonas spp. were found to be resistant to colistin in ICU samples. Enteric organisms were found to have high level ciprofloxacin resistant in 21.6% isolates, while S. paratyphi A isolation increased over a period of time. Yeast fungi isolated from blood predominantly were non-candida albicans (84.8%).

Neonatal Sepsis: Mortality and Morbidity in Neonatal Sepsis due to Multidrug-Resistant (MDR) Organisms: Part 1.

The major causes of emergence of multidrug-resistant organisms (MDRO) in neonatal sepsis include empiric antibiotic prescriptions, unregulated use of over-the-counter drugs, high incidence of healthcare associated infections (HAI), lack of awareness about antibiotic stewardship program and under staffing of neonatal intensive care units. In general, mortality due to MDRO sepsis is significantly higher as compared to non MDRO sepsis. Reported morbidities include prolonged use of total parenteral nutrition, need for central venous catheter, invasive ventilation, increased duration of hospital stay and neurologic sequelae.

Correction to: Neonatal Sepsis: Mortality and Morbidity in Neonatal Sepsis due to Multidrug-Resistant (MDR) Organisms: Part 1.

The article Neonatal Sepsis: Mortality and Morbidity in Neonatal Sepsis due to Multidrug-Resistant (MDR) Organisms: Part 1, written by Chand Wattal, Neelam Kler, J. K. Oberoi, Anurag Fursule, Anup Kumar and Anup Thakur, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 11 December 2019 with open access.

Prescription auditing and antimicrobial resistance at a tertiary care hospital in New Delhi, India.

This paper reports the antibiotic consumption data of Sir Ganga Ram Hospital, New Delhi and bacterial resistance over a seven-year period. Cephalosporins, penicillins and fluoroquinolones were the most widely prescribed antibiotics. A correlation was seen between Escherichia coli resistance to third-generation cephalosporins and increased cephalosporin use, as well as resistance to co-amoxyclav and its use.

Comparative evaluation of two rapid Salmonella-IgM tests and blood culture in the diagnosis of enteric fever.

PURPOSE: Enteric fever is a major public health problem in developing countries like India. An early and accurate diagnosis is necessary for a prompt and effective treatment. We have evaluated the diagnostic accuracy of two Rapid Salmonella-IgM tests (Typhidot-IgM and Enteroscreen-IgM) as compared to blood culture in rapid and early diagnosis of enteric fever. MATERIALS AND METHODS: A total of 2,699 patients' serum samples were tested by Rapid Salmonella-IgM tests and blood culture. Patients were divided into two groups. Test group - patients with enteric fever and blood culture positives for Salmonella Typhi; and three types of Controls, i.e. patients with non-enteric fever illnesses, normal healthy controls and patients positive for S. Paratyphi- A. In addition to this we have also evaluated the significance of positive Salmonella-IgM tests among blood culture-negative cases. RESULTS: The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Typhidot-IgM test and Enteroscreen-IgM test considering blood culture as gold standard were 97.29% and 88.13%, 97.40% and 87.83%, 98.18% and 92.03%, 96.15% and 82.27%, respectively. Typhidot-IgM test was found to be significantly more sensitive and specific as compared to Enteroscreen-IgM. Among blood culture-negative patients, Rapid Salmonella-IgM tests detected 72.25% additional cases of enteric fever. Although the Rapid Salmonella-IgM tests are meant to diagnose S. Typhi only, but these tests detect S. Paratyphi- A also. Thirty-eight patients who were blood culture-positive for S. Paratyphi- A were also positive by Rapid Salmonella-IgM tests. CONCLUSION: Rapid Salmonella-IgM tests offer an advantage of increased sensitivity, rapidity, early diagnosis and simplicity over blood culture.

Cutaneous cryptococcosis.

We report a case of an apparently immunocompetent male, who presented with a painless nodule over the upper abdominal wall. He gave a history of exposure to pigeon droppings. Cryptococcus neoformans was isolated from the lesion and no underlying disorder could be detected. He improved on treatment with oral Fluoconazole.

Variables affecting the performance of galactomannan assay in high-risk patients at a tertiary care centre in India.

BACKGROUND: Diagnosis of invasive aspergillosis (IA) in immunocompromised patients using galactomannan ELISA (GM-ELISA) has shown variable sensitivity and specificity. OBJECTIVES: To assess the diagnostic performance of GM-ELISA and analyze the effect of decreasing the cut off value, neutropenia, antifungals and piperacillin-tazobactam (PTZ). Prognostic value using 30 day all-cause mortality was also determined. MATERIALS AND METHODS: Serum samples from 81 patients categorized into "proven," "probable," and "possible," categories based on revised EORTC/MSG definitions were tested by GM-ELISA. RESULTS: Sensitivity of GM-ELISA in proven, probable and possible cases was 91.7%, 84.6% and 83.3% respectively. At an index cut-off value of 0.5 an increased sensitivity with minimal loss of specificity was observed. Use of antifungals demonstrated a decrease in sensitivity in proven and possible cases whereas it remained unaffected in probable category. Specificity increased from 75% to 100% with a positivity criterion of >2 consecutive samples. Although an increase in specificity was observed in patients not receiving PTZ, it was not statistically significant. Serial GM index values increased significantly in neutropenic patients and were associated with a poor prognosis. CONCLUSIONS: GM-ELISA may be a useful diagnostic and prognostic modality for the detection of IA in high risk patients.

Utility of GenoType MTBDRplus assay in rapid diagnosis of multidrug resistant tuberculosis at a tertiary care centre in India.

PURPOSE: Molecular methods which allow rapid detection of tuberculosis as well as drug resistance directly from clinical samples have become the most popular diagnostic methodology with the emergence of multidrug resistant tuberculosis. The aim of the present study was to evaluate the performance of a line probe assay, GenoType MTBDRplus for the rapid detection of Mycobacterium tuberculosis and mutations causing rifampicin and INH resistance directly in smear positive pulmonary specimens and also in M. tuberculosis isolates grown from various clinical specimens. MATERIALS AND METHODS: The MTBDRplus assay was done directly on 37 smear positive pulmonary specimens and also on 69 M. tuberculosis isolates obtained by rapid automated culture using Bact/Alert 3D. The results were compared with phenotypic drug susceptibility testing (1% proportion method) using Bact/Alert 3D. RESULTS: The sensitivity and specificity for detection of resistance to rifampicin was 100% and 97.3%, and to INH was 91.9% and 98.4%, respectively, in comparison with the phenotypic drug susceptibility testing. CONCLUSION: MTBDRplus assay had good sensitivity and specificity with turn around time of less than 48 hours. It may be a useful tool for rapid detection of multidrug resistant tuberculosis at a tertiary care centre.

High level ciprofloxacin resistance in Salmonella enterica isolated from blood.

PURPOSE: Over the last few years, resistance to ciprofloxacin in Salmonella enterica has become a global concern. The present study was undertaken to find out the susceptibility pattern of Salmonella enterica isolates in our hospital. METHODS: Blood cultures were done using BacT/ALERT 3D system. The antimicrobial susceptibility testing was carried out by the Kirby-Bauer disc diffusion method using CLSI breakpoints. Minimum inhibitory concentration was determined for ciprofloxacin-resistant strains using E-test and Vitek-1 automated system. RESULTS: A total of 25,953 samples of blood culture yielded 431 Salmonella enterica serotype Typhi and 198 serotype Paratyphi A isolates. Twenty-two isolates of serotype Typhi were resistant to ciprofloxacin, while two isolates of Typhi and two Paratyphi A were intermediately susceptible to ciprofloxacin. Ciprofloxacin resistance is 5.6% (24 isolates) among Salmonella enterica serotype Typhi. Ampicillin, chloramphenicol and co-trimoxazole resistance in Salmonella enterica serotype Typhi appears to have decreased to 14.9% (64/431) in comparison to the 27% (55/205) during 2003. All isolates were sensitive to ceftriaxone. CONCLUSIONS: Ciprofloxacin can no longer be considered as the drug of choice in treating Salmonella infections. While first-line antimicrobials may still have a role to play in the treatment of enteric fever, ceftriaxone remains the sole defence against ciprofloxacin-resistant Salmonella infections.

Nasopharyngeal carriage of Streptococcus pneumoniae.

OBJECTIVE: To determine nasopharyngeal carriage rate and prevalent serogroups/types (SGT) of S. pneumoniae in healthy children, assess their antimicrobial susceptibility and its implications over the heptavalent pneumococcal conjugate vaccine. METHODS: 200 healthy children aged between 3 months and 3 years attending Pediatric OPD at Sir Ganga Ram Hospital, New Delhi were studied. A nasopharyngeal swab was collected from each child which was processed to isolate Streptococcus pneumoniae. Serotyping was performed by the Quellung reaction. Antimicrobial susceptibility patterns were determined by disk diffusion and E test methods. RESULTS: S. pneumoniae carriage rate was 6.5%. Isolates belonged to serotypes 1, 6, 14 and 19, of which serotype 19 was the most common. None of the strains were totally resistant to penicillin though 2 (15.4%) were intermediately resistant. Overall, 84.6% of the isolates belonged to the strains covered by the heptavalent pneumococcal vaccine. CONCLUSION: The heptavalent conjugate vaccine covers most isolated strains, but since the number of strains is very small, it is suggested that there is need for further studies in different regions to assess the usefulness of this vaccine.

The pp67 mRNA assay in treatment and monitoring of cytomegalovirus disease in renal transplant patients in India.

The present report describes use of nucleic acid sequence-based amplification (NASBA) technology to detect pp67 mRNA of cytomegalovirus (CMV) in transplant patients in India. In our experience, pp67 mRNA assay was an accurate, rapid, and effective diagnostic tool to detect active CMV disease in 40.7% (50/123) of symptomatic transplant cases. This assay also allowed us to monitor CMV therapy. As part of the immunosuppressive regimen mycophenolate mofetil was found to increase the risk of developing CMV disease. All positive cases with this assay were subjected to antiviral therapy, with complete remission of the disease. At our center CMV NASBA assay has become the gold standard for the diagnosis of CMV disease in transplant patients.